Fig 2: Increased oxidative stress, upregulation of autophagy, and activation of DDR in adult SAMP8. a, d, f Representative Western blot analysis using antibodies against Nrf2, SOD2, p66Shc, p-p66Shc, p-Beclin1, Rab7 p62, p-Chk2, p53, p-p53, and β-actin in whole cochlear extracts. b, c Histograms representing the levels of Nrf2, SOD2, p66Shc, and p-p66Shc in SAMR1 and SAMP8 mice aged 1, 6, and 12 months (n = 16 cochleae per strain and per age) (*P ≤ 0.05, **P = 0.01, ***P ≤ 0.001 vs. 1 month age; #P ≤ 0.042, ##P ≤ 0.01, ###P ≤ 0.001 vs. SAMR1 of the same age). e Histograms representing the levels of p-Beclin1, Rab7, and p62 in SAMR1 and SAMP8 mice aged 1, 6, and 12 months (n = 16 cochleae per strain and per age) (*P = 0.024, **P ≤ 0.01, ***P ≤ 0.001 vs. 1 month age; #P = 0.022, ##P = 0.0036, ###P ≤ 0.001 vs. SAMR1 of the same age). g Histograms representing the levels of p-Chk2, p53, and p-p53 in SAMR1 and SAMP8 mice aged 1, 6, and 12 months (n = 16 cochleae per strain and per age) (*P ≤ 0.043, **P ≤ 0.01, ***P ≤ 0.001 vs. 1 month age; #P ≤ 0.025, ##P ≤ 0.01, ###P ≤ 0.001 vs. SAMR1 of the same age). β-Actin served as a loading control. Data are expressed as mean ± SEM. One-way ANOVA test followed by post hoc Tukey’s test. All experiments were performed in triplicate

Fig 3: Inhibition of PARP1-induced cell cycle arrest at the G2/M checkpoint. (A) Cell cycle analysis of TE6 and TE9 cells transfected with siPARP1 (n=3, cells transfected with siPARP1 were compared with the NC for each cell cycle). Compared with NC, siPARP1 significantly increased the ratio of cells in the G2/M phase and significantly decreased the ratio of cells in the G0/G1 phase. (B) Schema of ATM-Chk2-cdc25c pathways and cdc2/cyclin B1. (C) Western blot analysis showed that inhibition of PARP1 induced cell cycle arrest at the G2/M checkpoint through the ATM-Chk2-cdc25c pathway. PARP, poly (ADP-ribose) polymerase-1; siPARP1, small interfering RNA against PARP1; NC, negative control; KD, knock-down; cdc, cell division control; ATM, ataxia telangiectasia mutated; Chk2, checkpoint kinase 2.

Fig 4: Checkpoint activation and DNA damage sensitivity of SLX4-, MUS81-, and GEN1-depleted cells. (A) Analysis of phosphorylated CHK1, CHK2, and RPA32 in whole-cell extracts prepared from undamaged HeLa cells treated with the indicated siRNAs. Controls: Cells were treated with the indicated concentrations of HU for 24 h. (B) HU sensitivity of the indicated siRNA-treated cells. Error bars indicate ±SEM. (C) Quantification of early, mid-, and late S-phase populations (boxed) in asynchronous cultures of BrdU-labeled cells treated with the indicated siRNAs.

Fig 5: Interference of HR through a chemical inhibitor of Rad51 causes substantial toxicity in TMZ-resistant glioblastoma cells. (A) Intrinsically TMZ-resistant patient-derived IN-GB-2 cells were exposed to the indicated concentrations of TMZ or B02. After six days of incubation, cells were counted and cell survival was assessed by cell counting. Percentages of surviving cells were calculated in proportion to the cells treated with the vehicle (DMSO). (B) Parental TMZ-sensitive IN-GB-10 cells were exposed to increasing concentration of TMZ (10–320 µM) for three months. Two different clones (IN-GB-10-R1 and IN-GB-10-R2) growing in the presence of 320 µM of TMZ were isolated. Cells were treated with the indicated concentrations of TMZ and/or B02, and percentages of survival were calculated as described in A. *p < 0.1; **p < 0.001; Student’s t test. (C) U251-MG cells were exposed to increasing concentration of TMZ (10–320 µM) in a period of three months. One clone propagating in 320 µM of TMZ was isolated and cultured in the presence of TMZ for at least a month prior to the study. The parental (U251-MG-P) and resistant cells (U251-MG-R) were exposed to TMZ, B02, or combinations of TMZ and B02 at the indicated concentrations, and clonogenic cell survival assays were performed. Colonies were counted after seven days, and the percentage of survival was calculated in proportion to the control cells. **p < 0.05; Student’s t test (combination versus single treatments). (D) U251-MG cells were exposed to TMZ (200 µM), B02 (20 µM), or the combination of TMZ (200 µM) and B02 (20 µM) for 24 h. The representative histograms of the cell cycle analyses and quantification of the cell cycle stages are shown. (E) Intact Ape1/Chk2 signaling is required for recruitment of key HR proteins such as BRCA1 and Rad51 to facilitate HR, possibly through Ape1-mediated 3′ DNA-end processing of double-strand breaks. When Ape1 is deficient, simple base lesions are converted to complex clustered DNA lesions, which is further augmented after exposure to DNA DSB damages. Any possible failure in Ape1-mediated DNA-end processing results in impaired BRCA1 recruitment to damage sites, by which error-prone NHEJ becomes predominant repair pathway in Ape1-deficient cells, causing chromosome fusions and cell death. While deficiency of Chk2 causes replication defects and cell death, shortage of Ape1 and Chk2 is likely to create a new platform for alternative DNA end-processing factors that facilitate recruitment of HR proteins, increasing HR activity and thereby adaptation to constant genomic challenges.